Method of developing an immunogenic response using psoralen inactivated hiv

ABSTRACT

A method of developing an immune response to HIV using a composition of inactivated HIV. Inactivation is through psoralen and ultraviolet radiation. The composition is rendered more effective by the removal of structural features of HIV that interfere with immune response. In particular, sialic acid is removed to enhance immune recognition of the composition and to impair Complement Factor H. 
     A method of developing an immune response to HIV using a composition of inactivated HIV. Inactivation is through psoralen and ultraviolet radiation. The composition is rendered more effective by the removal of structural features of HIV that interfere with immune response. In particular, sialic acid is removed to enhance immune recognition of the composition and to impair Complement Factor H binding. CD55 and CD59 are also removed to prevent the binding of Complement Factor H.

CROSS REFERENCE TO RELATED APPLICATIONS

The present Divisional Application claims priority from co-pending U.S.patent application Ser. No. 10/971,445 filed Oct. 22, 2004 which claimspriority to U.S. Provisional Application Ser. No. 60/513,827 filed Oct.23, 2003; which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the fields of virology and immunology.Particularly, but not exclusively, it relates to a method of inducing animmune response to HIV using a psoralen inactivated composition of HIVand a substance for achieving the same.

2. Description of the Related Art

Human Immunodeficiency Virus

Human Immunodeficiency Virus (HIV) is a retrovirus within the slow orLentivirus group, and is the cause of Acquired Immunodeficiency Syndrome(AIDS). Some retroviruses that attack the immune system, such as HIV-1,are variable and mutate readily, creating many strains of varyinggenetic composition that hamper efforts to develop effective treatment.These strains, which may be categorized into groups or subtypes, haveindividual biological characteristics. Sequences within a subtype mayhave genetic clustering or similarities that sometimes reveal theircommon lineage. However, variations in evolutionary rate can producedifferences among mutations even within a subtype. Further, the tendencyof retroviruses to recombine with related retroviruses complicate theviral genotype.

HIV uses its RNA as a template for making complementary viral DNA intarget cells through reverse transcription, viral DNA can then integrateinto the DNA of an infected host. HIV infects cells having surface CD4,such as lymphocytes, monocytes, dendritic cells and macrophages, anddestroys CD4 positive helper T lymphocytes. This process relies in parton two glycoproteins of HIV. These glycoproteins are gp120 (an Envglycoprotein, the exterior receptor-binding component) and anon-covalently interacting partner, gp41 (the Env transmembraneglycoprotein.) Gp120 and gp41 are associated in a trimeric unit, wherethree molecules of gp120 are exposed on the virion surface and areassociated with three molecules of gp41 in the viral lipid membrane.Gp120 binds to a CD4 receptor on the surface of helper T cells. Thisbinding is generally considered to be high affinity, and can be furtherenhanced by high sialic acid content on the surface of the virus; sialicacid reduces the threshold binding energy needed to overcome repulsiveelectrostatic forces. The virus then begins to fuse with the T cell,producing structural or conformational changes and exposing otherreceptors. Upon fusion, the gp120 fragment is shed, exposing the gp41ectodomain in a process that also varies conformationally. Gp41 is thenavailable to project peptide fusion domains for binding to the targetcell. This leads to HIV entering and infecting the target cell.

The envelope of HIV begins formation from the plasma membrane of thehost cell when the virus buds through the cell membrane. Thus, theenvelope includes the lipid and protein constituents of the host cell.(Frank, Ines, Heribert Stoiber, et al., Vol. 10, pp. 1611-20 (1996))(Stoiber, Heribert, et al., Vol. 15, pp. 649-74 (1997)) Some envelopedviruses use spike proteins to mimic the host molecules in order to bindto target cell receptors and to enter other target cells. However, thesespikes can also be antigenic surfaces for immune system recognition. YetHIV offers protection. In addition to the variability of conformationalchanges, gp120 provides other surface features that disguise it fromimmune detection and attack, such as a coating of glycoproteins,covalently bound sialic acid residues, or steric occlusion. (Haurum,John, Treffen Thiel, et al., Vol. 7(10), pp. 1307-13 (October 1993))(Sande, Merle, et al., The Medical Management of Aids, (6th ed. 1999))(Cohen, P. T., The AIDS Knowledge Base, (3rd ed. February 1999))

The core of the HIV virion functions as a command center. Inside an HIVvirion is a capsid composed of the viral protein, p24 (CA). The capsidholds two single strands of RNA, each strand of which provides a copy ofHIV's nine genes, which encode 15 proteins. Of the nine genes, three(gag, pol and env) are considered essential. Six additional genes arealso found within the 9-kilobase pair RNA genome (vif, vpu, vpr, tat,rev and nef) More specifically, the env gene holds the information orcode for creation of gp160, which breaks down into gp120 and gp41.Likewise the gag gene encodes the matrix (p17 or MA), capsid (p24 orCA), nucleocapsid (p9 or NC) and p6. The pol gene provides the geneticinformation for the virus to produce the reverse transcriptase enzyme aswell as the integrase enzyme and RNAse H enzyme. The other six genes areregulatory, and control the mechanisms of infection and replication(vif, vpu, vpr, tat, rev and nef). Among other things, the nef geneholds information for efficient replication, while vpu holds informationregulating the release of new viral particles from the infected hostcell. Ultimately, in order for HIV to infect a target cell, it mustinject the HIV genetic material into the target cells cytoplasm.

As noted above, the nef gene is believed to aid efficient replication ofHIV. The creation of a new virus particle occurs at the host cell'smembrane. Nef appears to affect an infected cell's environment in a waythat optimizes replication. Viral proteins collect near the host cellsmembrane, bud out within the membrane, and break away. These proteinsare the three structural proteins (gp160, gp120, gp41) plus two otherinternal precursor polyproteins (Gag and the Gag-Pol). The Gag-Polprotein brings two strands of the positive RNA into the bud, whileprotease cuts itself free. After the virus has budded, protease cutsitself free and cuts up the rest of the proteins in Gag or Gag-Pol,releasing the various structural proteins and reverse transcriptase. Theviral proteins are not functional until they are separated by theprotease. Thus, protease is responsible for cleavage of Gag-Pol and thesmaller Gag polyprotein into structural proteins. Released proteins p24,p7 and p6 form a new capsid, while at the base of the lipid membrane isp24. In this process, gp160 breaks down into gp120 and gp41 by a hostenzyme.

Most HIV vaccines use portions of the envelopes of these glycoproteins(gp160, gp120, and gp41) in an attempt to induce production ofneutralizing antibodies against the envelope spikes of the virus.(Johnston, et al., 2001) Some have been successful in producing hightiters of neutralizing antibodies. The thought behind this approach isthat the antibodies that bind to these glycoproteins would neutralizethe virus and prevent infection. A functioning immune system could thenactivate the complement system, which would cascade to lysis and destroythe virus. The complement system is a series of circulating proteinsthat “complements” the role of antibodies. The components of thecomplement system are activated in sequence or turn, which is thecomplement cascade. The conclusion of complement is a protein complex,the Membrane Attack Complex (MAC) that seeks to attach to an invadingorganism's surface and to destroy it by puncturing its cell membrane.

However, HIV provides an additional protection against humoral immuneresponse. HIV will activate human complement systems even in the absenceof specific antibodies. This activation would be harmful to the virus ifcomplement were left unimpeded to reach MAC, triggering virolysis.However, HIV avoids virolysis by incorporating into its structurevarious molecules (e.g., CD55, CD59) that regulate complement. HIVincludes these cell membrane molecules in the virus membrane uponbudding from infected cells, or by attachment to gp41 and gp120.Complement Factor H may be incorporated into the structures of both gp41and gp120. Factor H inhibits the activity of C3b, a molecule that iscentral within complement cascade. This interaction with complementcomponents enables HIV to take advantage of complement activation toenhance infectivity, follicular localization, and target cell range.

Vaccine Therapy and Related Art

Immunotherapy involves the use or stimulation of the immune system withrespect to a condition or sensitivity. Vaccines are a form ofimmunotherapy. In 1955, Dr. Salk introduced the poliovirus vaccine; thisvaccine used the chemical formaldehyde (formalin) to kill the virus orrender it non-infective or inactive, so that it could be administered topatients. In 1961 Dr. Sabin introduced a live attenuated relativelyavirulent poliovirus vaccine. The Sabin vaccine was basically composedof viral mutants capable of eliciting an immune response but not capableof significant active replication or virulence, and therefore wereconsidered relatively safe for human use.

There have been effective vaccines against retroviruses in animals. Onevaccine is available for feline immunodeficiency retrovirus (or FIV)(i.e., Fel-O-Vax); a second example is a vaccine against EquineInfectious Anemia Virus (or EIAV), (i.e., EIAV(UK)deltaS2) an importantretroviral infection of horses. These vaccines argue powerfully thatvaccines can work against retroviruses, although neither disease is anideal model for HIV in humans. (Beyer, 2003)

However, a vaccine for HIV has proven elusive. The vast majority ofvaccines under consideration, research, or trials are comprised ofeither “live” attenuated viral particles or whole inactivated viralparticles. The use and research of recombinant technology, adenoviralvectors, DNA-based vaccines or a combination thereof has tested theboundaries of immunology, offering some hopes for addressing HIV. Suchimmunogenic compositions could be used for the following purposes:

To enhance the immune system of a person who has already been infectedwith the disease systemically.

To prevent a person from contracting the disease after exposure.

To prevent a person from contracting the disease prior to exposure. Thisis the most common use for a vaccine today.

To prevent a patient from contracting a different strain of HIV disease,particularly non-compliant or immunosuppressed patients.

To prevent vertical transmission from mother to fetus or from mother tonewborn.

To attenuate HIV disease in an HIV negative patient who contracts thedisease at a later date

To research potential compositions and methods for any of the purposesabove

Unfortunately, medicine lacks a definition for HIV immunity. (Gonsalves,Gregg, Basic Science (2000)) (Cohen, 1999) This is a fundamental problemwith an important consequence: there is no known correlate of protectionagainst HIV. However, there are well-characterized correlates fordisease progression, such as viral loads and CD4 counts. Furthermore,there is no evidence that any of the current candidate vaccines canelicit responses in HIV-positive patients that would improve theseparameters (viral loads and CD4 counts) for an extended period. (Beyrer,Chris, “The HIV/AIDS Vaccine Research: An Update.” The Hopkins Report(January 2003)) Additionally, while there have been advances in someanimal models, there is no validated animal model system for testingvaccine candidates, an obvious limitation when working with a highfatality pathogen such as HIV. (Beyrer, 2003) Current life expectancyafter contracting HIV disease is approximately 10 to 15 years. Even avaccine that failed to prevent transmission but extended life expectancyof a patient after contracting the disease would constitute animprovement.

Inactivated viruses may be useful for research and medicine. In fact,most of the successful early vaccines relied on inactivated virus.Inactivation produces a virus that is not infective, yet still inducesan immune response based on its residual characteristics. An inactivatedvirus is typically generated from stocks of a virulent strain grown incultured cells or animals. A potentially virulent virus is then madenon-infectious or inactivated by chemical treatment. Viruses are bydefinition non-viable entities; they do not consume oxygen and food, nordo they produce waste; they replicate via their host, as described abovefor HIV. Viruses have no inherent metabolic activity and do not produceadenosine triphosphate (ATP). However, a live virus vaccine is capableof reproduction, while a killed virus vaccine is not. In general, livevaccines are more efficacious but also more dangerous than killedvaccines.

When a virus is inactivated, an immunogenic composition based oninactivated virus must retain its antigenicity in order to be useful.The inactivation process should preserve the three-dimensional structureof the virus while at the same time eliminating its virulence. Manymethods are available to inactivate or kill a virus, but most destroy orchange the three-dimensional structure of the virion, harming itsantigenic characteristics. Originally, formaldehyde (formalin) treatmentwas used; for example, the Salk poliovirus vaccine was aformalin-inactivated preparation of three virus serotypes. Despite itswide use in early vaccines, formalin is difficult to remove andtherefore poses the danger of residual toxicity. More recently,β-propriolactone has become a commonly used chemical to inactive a virusbecause residual amounts of the reagent can be readily hydrolyzed intonon-toxic products. U.S. Pat. No. 4,169,204 to J. Hearst, et al.,suggested the use of psoralens with irradiation to inactivate virusesfor vaccine preparation. Psoralens are attractive because of theirability to inactivate virus without damaging the structure and withoutharmful residue. (Hanson, C. V., Bloodcells, Vol. 18(1), pp. 7-25(1992)) Psoralens occur naturally in plants, including limes and celery,which use them to attack insects and fungi.

As noted above, the general notion of using psoralen to inactivateviruses is known. For example, U.S. Pat. No. 5,106,619 disclosedpsoralen inactivation of a live virus in order to prepare vaccines. Thatinvention involved treatment or inactivation of virions usingfurocoumarins, including 4′-aminomethyl-4,5′, 8-trimethylpsoralenhydrochloride (AMT), and ultraviolet light in a limited oxygenenvironment. The inactivation is directed to double and single strandedDNA viruses, double and single stranded RNA viruses, and enveloped andnon-enveloped viruses. This disclosure was general, and did notspecifically contemplate HIV.

Some inventors have contemplated the use of psoralen in an HIV vaccineor composition. U.S. Pat. No. 6,107,543 disclosed a whole particle HIVimmunogen that is inactivated preferably by gamma radiation; alsodisclosed, however, are a variety of alternative inactivation methodsincluding psoralen, formalin, β-propriolactone, etc. The whole particleis treated for removal of the outer envelope proteins gp120 or gp160,while retaining the remainder of the structure. An alternativeembodiment is a reduced immunogen comprising the remaining purified geneproducts, such as those encoded by the gag genes, the pol genes, thetrans-membrane protein gp41, or the remaining genes of the HIV genome.

U.S. Pat. Nos. 6,383,806 and 6,503,753 disclosed a composition andmethod for development of an HIV vaccine based on psoralenphotoinactivation of Reverse Transcriptase (RT). In other words, theobjective of this invention is to promote an immune response based onthe inactivation of a single inactivated enzyme within HIV. Preservationof the remainder of the particle is deemed to enhance immune response tothe composition.

Although psoralen has been contemplated by inventors for use in an HIVimmunogen or vaccine, none have looked to certain structuralpreservation issues inherent with psoralen inactivation of HIV. Forexample, HIV is highly mutagenic, changing structures frequently in theprocess of reverse transcription. Mutation may provide a means for anHIV strain to escape immune response caused by a vaccine. (Cohen, 1999)In addition, the preservation of HIV structure may result in thepreservation of HIV components that disadvantage immune response.

Past efforts have not focused on the problems of mutation. HIV is ahighly mutagenic retrovirus which, through reverse transcriptaseconverts its RNA into DNA. HIV reverse transcriptase is error prone,leading to mutation. Further, rapid replication exacerbates mutation.The high level of genomic diversity in HIV complicates diagnosis,treatment, and public health monitoring of disease progression. Inparticular, this diversity is manifested in biological peculiaritiescharacterizing as infectivity, transmissibility, and immunogenicity. Thedivergence in viral genotypes of HIV has contributed to polymorphism,transmission efficiency, and the historical epidemic development of HIV.The variety of subtypes and sub-subtypes with each having a peculiarthree dimensional structure can render a subtype vaccine ineffective fora patient having a different subtype. The high rate of mutation of HIVis certain to complicate selection of the appropriate immunogen.

The preservation of HIV structural components may present performanceissues. As with U.S. Pat. No. 5,106,619, both U.S. Pat. Nos. 6,383,806and 6,503,753 preserve whole particles. The later inventions aredirected to inactivating only the RT. The preservation of the antigenicstructure is intended to take advantage of a wider range of immunogens.This preservation of the correct antigenic conformation is consideredimportant for access to the cytoplasm via micropinocytosis ormannose-receptor mediated uptake at dendritic cells. U.S. Pat. No.6,107,543 includes psoralen inactivation within its disclosed method,but conversely required the removal of envelope glycoproteins gp120 andgp160 (but not gp41) because antibodies to those glycoproteins mightfacilitate virus absorption to cells. In fact, it is known that HIV canbind to and use C3b as ligands to permit infectious immune complexes tobind to dendritic cells and B lymphocytes. Antibodies to gp160 or gp120sometimes lead to concentrations of virus in the lymph nodes and spleen.The '543 approach, like the others, would preserve transmembrane proteingp41 and some or all of the viral membrane.

At any rate, this preserved viral structure can hold unintendedconsequences. First, as described above gp160, gp120, and gp41 providebinding sites for complement factor H. (Pinter, Claudia et al, AidsResearch and Human Retroviruses, Vol. 11(5), pp. 577-88 (1995) (Pinter,Claudia, et al., Aids Research and Human Retroviruses, Vol. 11(8)(1995))(Stoiber, Heribert, et al., Immunobiology, Vol. 193, pp 98-113(1995)) Accordingly, the retention of these structures means that factorH will interfere with humoral immune response following vaccination. Theremoval of gp120 gp160 in U.S. Pat. No. 6,107,543 may mitigate thiseffect to some degree; nevertheless, the preservation of the gp41 FactorH binding sites would work against the immunogenicity of thecomposition. Second, both approaches are silent as to the cellularplasma membrane and retain some or all of the viral membrane, includingcertain bound proteins that interfere with immune response. As anassembling, replicating HIV particle buds through the infected cellplasma membrane, the membrane is enriched by CD55 (the decayaccelerating factor) and CD59 (the homologous restriction factor) thatregulate complement. These molecules are incorporated into the viralmembrane upon budding from infected cells. Preservation of some or allof these features or structures could interfere with complementactivation and humoral response. (Saifuddin, 1995) Third, HIV surfacecomponents bear sialic acid, which could remain on the preservedstructure of inactivated HIV. Sialic acids are typically found on hostproteins and cellular structures; high sialic acid content on a virus,even if the virus were inactivated, would limit the host's ability torecognize the virus and respond properly. Importantly, sialic acidresidues are also used in the binding of Factor H. (Meri, Seppo, et al.,“Discrimination Between Activator and Nonactivators of the AlternativePathway of complement Regulation: Regulation Via a Sialic acid/Polyanionbinding site on Factor H.” Proc. Natl. Acad. Sci., USA, Vol. 87(10), pp.3982-6 (May 1990)) (Blackmore, T. K., et al., J. of Immunology, Vol.157(12), pp. 5422-7 (December 1997)) (Kuhn, S., et al., Eur. J.Immunol., Vol. 26(10), pp. 2383-7 (October 1996)) (Pangburn, M. K., etal., J. of Immunology, Vol. 164(9) (May 2000))

The present invention is directed to an immunogenic composition thataddresses these issues. It is intended that by creating compoundsavailable to target different subtypes and aspects of HIV, it willadvance treatment and research. Ultimately, it is hoped to extendsurvival and to improve the quality of life for infected individuals.

SUMMARY OF THE INVENTION

HIV is among the viruses sensitive to inactivation by psoralen. Theviral structure is preserved during the process of psoraleninactivation. This preservation is advantageous for the production ofbroad based antigenic response. In the present invention, theinactivated viral structure is modified to remove or neutralize selectedfeatures that would interfere with immune response. In particular,sialic acid and certain binding sites for complement factor H areneutralized; in some cases the outer cellular plasma membrane may beremoved. The primary effect of this treatment is to obviate interferencewith immune humoral response by human complement factor H and otherregulators of complement activity (RCA). In addition, the removal ofsialic acid reveals to the immune system that viral components aredistinct from the host.

The present invention is tailored to the genotype of virus presented.HIV replicates rapidly and mutates readily through reverse transcriptaseand recombination. Each group, subtype, sub-subtype, and circulatingrecombinant form of HIV-1 and HIV-2 is structurally unique. Missing asubtype could pose serious consequences. For an immunogenic compositionto be effective, it must take into account not only interference bypreserved structure, but each viral group, subtype, sub-subtype, etc. ofconcern under the given circumstances. Thus, a vaccine may be tailoredto the viral strain(s) present in the host. Accordingly, the compositionmay preferably be generated from a genetically relevant sample; in thecase of a composition intended for the vaccine of an infected host, forexample, the relevant sample may be drawn from or matched to the hostperson. In the case of a composition intended for the vaccine of anuninfected human or other animal, then the relevant sample may be basedon a probabilistic assessment of the risk of exposure for that human orother animal.

Accordingly, the present invention is a composition capable of invokingan immune response, wherein certain predetermined strains of HIVrelevant to the use of the composition are isolated and inactivatedusing psoralen and exposure to ultraviolet light. The present inventionis further characterized by the removal of certain features from theinactivated HIV that impair immune response. The composition may furthercomprise pharmacological carriers, stabilizers, or excipients.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows Psoralen formation of photoadducts with nucleic acid uponexposure to ultraviolet light.

FIG. 2 demonstrates how increasing polysaccharide length enhancesimmunogenicity up to a maximum of 16 monosaccharides.

FIG. 3 is a schematic drawing showing the chain structures of C3 and CVFand their relationship.

DESCRIPTION OF THE INVENTION

As noted above, the general use of psoralen for inactivating HIV toproduce an immunogenic composition or vaccine is known. However, themutability of HIV and its ability to interfere with immune response canimpair the performance of prior undertakings. The present invention isan immunogenic composition and method directed to the problems posed bypreserved features of viral structure, the high rate of HIV mutation,and HIV resistance to immune response. The present invention isimmunotherapeutic, in that it involves the use or stimulation of theimmune system with respect to a specific condition or sensitivity. Theimmune system of concern may be that of a human or any other animal,such as a chimpanzee or mouse. As used here, immunotherapy encompassesthe tailoring of an immunogenic composition to optimize performance inthe context of the highly mutable genotype combined with active viralinterference with immune response. It is intended that the presentinvention advance diagnostic procedures, analyses, and evoke an immuneresponse in animals. It is also contemplated that given the developmentof an effective immunogenic composition, a vaccine may be developed andadministered using the composition in order to produce immunoprotectivefactors within a patient.

Accordingly, one aspect of the present invention is a compositioncapable of invoking an immune response, wherein certain predeterminedstrains of HIV relevant to the use of the composition are isolated andinactivated using psoralen and exposure to light, and further wherefeatures of HIV that impair immune response are removed from theinactivated HIV. The composition may further comprise apharmacologically appropriate carrier, stabilizer, or excipient.

In an alternate embodiment intended for use as a vaccine for an infectedanimal, the vaccine may include those strains present in the animal atthe time the sample was drawn, matching the genotype of the vaccine tothat of the infection. Alternatively, a preventive vaccine may includestrains of concern based on probability of exposure. The vaccine may betreated to remove features of the HIV that impair immune response.

Another aspect of the invention is a method of preparing an immunogeniccomposition comprising determining the strains of HIV relevant to theuse of the composition, inactivating the strains using psoralen andexposure to light, and removing or modifying features of the HIV thatimpair immune response. In particular, these features or components maycomprise the complement Factor H binding sites for gp160, gp120, andgp41, removal of sialic acid residues, and optionally the outer cellularplasma membrane. Therefore, a methodology of the present inventioncomprises for preparation of an immunogenic composition includes:

1. Determining the strains of HIV that are of concern

2. Isolating and culturing the strains of concern

3. Separating the virus from the culture media

4. Optionally removing the cellular outer plasma membrane

5. Adding psoralen and a DNA-repair enzyme blocking agent

6. Irradiating with ultraviolet light

7. Removing or neutralizing CD55 and CD59

8. Desialation of the inactivated virus

The present invention is thus an immunogenic composition comprised of atailored combination of psoralen inactivated HIV subtypes, and a methodof preparing and using the same.

Determination of Relevant Strains

The determination of the strains of HIV will depend on the embodimentand application of the present invention. There are a wide variety ofapplications for immunogenic compositions; accordingly, the followingshould be construed as exemplary and not limiting. The determination ofHIV strains that are relevant may be considered in three exemplarycategories: (i) research related, development, analytical; (ii)therapeutic; and (iii) preventive.

In one category of embodiments, the immunogenic composition may becontemplated for use in research or clinical analysis. For research, thestrains of concern will be determined by the objectives of thescientific investigation. That is, the procurement, isolation, andculture of the HIV virus will accord with the investigatory design andobjectives. The determination of relevant strains for medical researchmay likely parallel that for any envisaged therapeutic or preventiveneed, such as vaccine development. Purely academic research mayencompass aspects such as the development of research tools or expandingknowledge about strains unrelated to the HIV epidemic. The immunogeniccomposition may also be useful in studies of immune response, viralevolution, epidemiology, and analysis of viral behavior.

In another category of embodiments, the immunogenic composition may beintended for administration to an HV infected human (or other animal orhost), possibly as a therapeutic vaccine. In general, multivalentimmunogenic compositions should have the ability to induce an immuneresponse against diverse viral isolates; in the past, this multivalencewould involve a combination of inactivated viral particles from avariety of strains of concern. However, HIV within an individual host israrely static, and continues to evolve through genetic mutation andrecombination. Past efforts to target common strains or variants in amultivalent HIV vaccine rely on macroscopic public health predictors andconservative over sampling of HIV strains. In the present invention, thestrains of HIV may be genotyped and isolated from a sample drawn fromthe infected human or other animal. That is, peripheral bloodmononuclear cells (PBMC) or other sample may be drawn in order toidentify the HIV present in the infected human or other animal. Anytyping method appropriate to the circumstances may be used, includingsequence based diagnostic genotyping, heteroduplex mobility assay (HMA),analyte specific reagents assay genotyping, molecular diagnostics, genedetection products, and DNA probe-based products. A vaccine may then becomposed of those strains present in the human at the time the samplewas drawn. Thus, in one embodiment, the HIV in a human (or animal model)is genotyped and the information is used to create a composite vaccinecomposed of stock components derived from inactivated virus for each ofthe identified strains. As described below, this alternative may involvea vaccine derived from virus cultured from human PBMCs in vitro or othersample drawn from a person (or other animal) that then undergoes theprocess of psoralen inactivaton before being re-introduced into thatperson or other animal as an immunogen. In this way, the immunotherapyis tailored to the genotype of that host's virus. The methodology of thepresent invention contemplates a plurality of samples to identify andaddress changes in strain dominance or composition over time and, inresponse, enable changes in treatment. When a dominant strain isdestroyed, a distinct but related resistant variant may then emerge,requiring further immunotherapy.

In another category of embodiments, the immunogenic composition may becontemplated for administration to an uninfected human, possibly as apreventive vaccine. In the past, the preventive composition andadministration have been similar to that used in therapeutic multivalentcompositions; such compositions may be based on the combined use ofviral particles from a wide variety of strains intended to induce abroad immune response. Such an approach is contemplated as oneembodiment of the present invention in preventive form.

Alternatively, the present invention in a preventive embodiment may alsobe based on a probabilistic assessment of the risk of exposure for thatperson. Notably, U.S. Pat. No. 6,503,753 contemplated personalpreventive vaccines in cases where the risk of transmission wasprimarily through a particular individual; accordingly, the inactivatedstrains may be derived from a sample drawn from that infectedindividual. Such personal vaccines could be used as an embodiment of thepresent invention. Likewise, laboratory workers or healthcareprofessionals may face heightened risk from occupational exposure tocertain known strains and may benefit from improved immune response tothose specific strains. Alternatively, strain determination for personalvaccines may be based on behavioral and demographic risk factors for HIVinfection in geographic areas of concern. Such analysis could considerbehavioral patterns in the context of historic, epidemic, and geographicdata supporting an exposure probability analysis. In geographic areaswhere the epidemic has spread beyond groups with identifiable riskfactors, then multivalent prevention could reflect all known strains bygeographic area of concern.

Procurement and Preparation for Irradiation

The following embodiments are to be considered exemplary and notlimiting, as there are well established methods for procurement,isolation and culturing viruses. In fact, the actual virus may beprocured in a variety of ways. Individuals infected with the HIV strainsmay be sampled and the virus may be isolated, purified, cultured, andtyped using procedures that are known in the art, and are in partdiscussed below. Such samples may be drawn from PBMC or other fluids,such as saliva, or tissues, such as relevant mucous membranes; however,as is known in the art, PBMC may be preferable for its inclusion ofimmune components, depending on the application. Alternatively, HIV maybe procured from existing samples of known commercial viral stocks orlaboratory isolates. Viral particles may also be produced bytransformation of cells with viral-encoding vectors, which is the uptakeof foreign genetic material into a cell. Transformation is usuallyaccomplished by physical means such as co-precipitation of the DNA withinsoluble calcium chloride. (Nicholls, Desmond, An Introduction ofGenetic Engineering (2nd ed. February 2002)) Transformed DNA can eitherexist in the cell as an episomal (extra chromosomal) element or beintegrated within the nuclear genome. The efficiency of a DNA transferinto cells depends on the particular method used. The descriptions belowmay be common to the various categories of embodiments herein, unlessotherwise specified.

The present invention contemplates various preferable culture media. Afirst preferable culture media is the PBMC drawn from an HIV infectedhost. In this embodiment, the HIV cells would replicate within thepreserved in vitro host environment. Such a media may be safer andsimpler for subsequent administration to the host. However, many culturemedia such as human cells or yeast include DNA-repair enzymes that canreverse the effect of psoralen. These enzymes can repair DNA and RNAthrough transcription coupled repair and global genomic repair. Thus,culturing in PBMC or yeast cells would be improved by inhibition of anyenzymes that would tend to repair the DNA and RNA following exposure topsoralen and ultraviolet light. In a preferred embodiment, such mediamay be treated with repair enzyme inhibitors such as novobiocin,aphidicolin, or both. (Brenneisen, Peter, et. al., J. of BiologicalChemistry, Vol. 275(6), pp. 4336-44 (Feb. 11, 2000)) (Niggli, H. J.,Mutation Research, Vol. 295(3), pp. 125-33 (August 1993))(Cleaver, J.E., J. of Cancer Research, Vol. 47(9), pp. 2393-6 (May 1987))(Rosentein, B. S., et al., Environmental Mutagenis, Vol. 8(3), pp.335-43 (1986)) In another preferred embodiment, the culture media is anymedia lacking or having low levels of such DNA-repair enzymes, such asFanconi anemia type C. Fanconi anemia type C has a deficiency ofDNA-repair enzymes, reducing recombination and making it preferable foruse with psoralen inactivation. Some mutant cultures are known to bedefective in excision repair of UV-induced pyrimidime dimers and arehypersensitive to photo addition of both mono and bi-functionalpsoralens. Examples are uvrA, B, or C mutants of escherichia coli, ofRAD3 type units of saccharomyces cerivisiae, Chinese hamster ovarycancer cells complementation groups 1 and 4, Xeroderma pigmentosumgroups A and D. Other culture media may be functionally equivalent solong as the operation of UV light damage DNA-repair enzymes isinhibited.

For an HIV negative human (or animal) a suitable vaccine/immunogen canbe produced by culturing the virus in vitro before psoraleninactivation. Many culture media are available but a preferredembodiment would be a human's own peripheral blood mononuclear cells(PBMCs). This would subject the virus to a selective pressure generatedby the human's own immune system. The strain(s) of HIV that replicate invitro in such a culture would be the strains the human would most likelyreplicate in vivo in producing an active infection. Therefore each HIVnegative and HIV positive human could have a tailored made vaccine.

Separation of the virus from culture cells may be accomplished bycentrifuging the cells for ten minutes at 2000 rpm and at 4° C. Thesupernatants may then be filtered twice through a 0.22 millimicronMillipore filter.

In some cases, it may be desirable to remove host cellular outermembrane or membrane particles that could impede access to the viralcomponents. In that case, disruption or removal of the outer membranecan be accomplished by means that are known in the art, such astreatment with ethanol, detergents, phenols, or other solvents.Importantly, removal of the outer membrane should not otherwise alterthe viral structure or denature any viral proteins. (Levinson, Warren,Medical Microbiology & Immunology: Examination and Board Review (7th ed.July 2002)) In a first embodiment, such removal may be accomplished bydetergents; detergents are “surface-active” agents composed of along-chain lipid soluble hydrophobic portion and a polar hydrophilicgroup. The hydrophilic group may be a cation, an amine, a non-ionicgroup or multivalent. Surfactants interact with the lipid in the cellouter membrane through their hydrophobic chain and with the surroundingwater through their polar group and thereby disorganizes the outermembrane. Quaternary ammonium compounds, such as benzalkonium chlorideare cation detergents widely used for skin antisepsis and would bepreferable for application here. Alternatively, ethanol will disorganizethe lipid structure in the outer membrane; uncontrolled, it coulddenature proteins—which may be a limiting factor. Ethanol is moreeffective when diluted with water, for example, 70 percent ethanol ispreferable to 100 percent. Thirdly, phenols such as hexachlorophene orcresol may be appropriate in some embodiments. The use of any of thesesubstances should be under conditions that preserve the antigenic natureof the inactivated virus, preserves the viral structure, and avoidsdenaturing of the viral proteins.

The inactivation agent is preferably psoralen, as it is able to preservethe antigenic properties of the viral structure while inactivating RNAand DNA. Psoralens are a class of photo-mutagenic andphoto-chemotherapeutic compounds that covalently modify nucleic acids.They belong to a family of small molecules that intercalate andphotoalkylate DNA and RNA (both single stranded and double stranded.)The primary targets of psoralens are thymidine residues in DNA anduracil residues in RNA; these molecules form both monoadducts andinterstrand cross links. The reaction takes place between the 3,4(pyrone) or 4′,5′ (furan) double bonds of the psoralen and the 5,6double bond in pyrimidines. The photobinding of psoralens to doublestrand DNA (dsDNA) results in substantial structural distortion of thenormal DNA double helix. (Spikes, John D., Photosensitization inMammalian Cells, Ch. 2 (1983)) (Averbeck, D., et al., Mutagenic Effectsof Psoralen-Induced Photo adducts and Their Repair in Eukaryotic Cells,Pp. 933-59 (1988))

The immunogenic composition for one or more predetermined subtypes ofHIV may be prepared for irradiation as follows. The HIV virions ofconcern in culture may be diluted in a solution containing4′-aminomethyl4, 5′, 8-trimethylpsoralen (AMT). Psoralen derivativesreadily pass through cell walls and through virus coats and can thenphotoreact after exposure to UV light with the nucleic acids inside,producing pyrimidine dimers. AMT cross-links the viral RNA and DNA. Allpsoralens are photoactive.

Irradiation

Upon exposure to UV light, the psoralens form photoadducts with nucleicacids. This process does not require oxygen, and preferably oxygenshould be removed. Psoralens can be irradiated before the addition ofnucleic acids and photoadducts will still result. The HIV virus can thenbe inactivated with AMT and UV light. The virus is then unable toreplicate its nucleic acid because the cross-linked DNA or RNA cannot becopied. The virus is thereby “killed” by inactivation of its DNA or RNA.Because photoreaction blocks the replication and expression of nucleicacid, it forms the basis of a technique for eliminating the infectivityand virulence of viruses. The surface of the virus remains largelyunmodified, rendering the inactivated virus potentially useful forvaccines.

Psoralen photo-inactivation has proved to be superior to conventionalinactivation techniques, such as formaldehyde treatment, for thepreservation of antigenicity and immunogenicity in experimentalinactivated vaccines. (Hanson (1992)) Photoreaction with AMT has beenshown to remove the HIV infectivity from HIV-infected cells withoutaltering antigenic surface properties; these inactivated cells retainnormal reactivity with monoclonal antibodies to a variety of bothcellular and viral antigens. (Hanson, 1992)

In HIV infected cell cultures, numerous DNA transcripts of the viralgenome are produced in the infected cell and may eventually spill fromlysing cells into the culture medium. While it is not known if such DNAor RNA poses a hazard of transfection or transformation, the psoralenphotoreaction would be expected to inactivate this free DNA and ifanything even more rapidly than it inactivates the RNA-containingvirions, thus providing a safety factor not available from conventionalinactivating techniques such as heat, alcohol or detergent. Furthermoreheat, alcohol or detergent cannot evenly inactivate the RNA and DNA ofviruses. (Hanson, 1992)

The kinetic mathematics of psoralen inactivation is frequently nonlinearand can show a “tailing off” effect. This tailing off probably resultsfrom loss of psoralen activity during the virus irradiation due tophotodegradation of the psoralen itself. Preferably, periodic additionof psoralen during virus inactivation will maintain linear kinetics. Theloss of psoralen activity parallels viral inactivation. Two successivesteps of irradiation are preferable for effectiveness and for creatinghigher safety margins for the vaccine. For example, a first exposure of405 nm wavelength followed by a 365 nm wavelength exposure is preferablefor a higher production of cross links capable of disabling a DNA or RNAmolecule. In embodiments where a single exposure is required, then a 365nm wavelength exposure is preferable. The dose rate shall be appropriatefor the psoralen absorption under the circumstances; excess dose ratebeyond viral inactivation may lead to photodegradation of viralproteins.

Preferably, a sample of the irradiated viral cells may then be culturedand analyzed to ensure inactivation and to eliminate the possibility ofresidual infectivity.

Treatment of Structural Features that Could Impair Immune Response

Regulators of complement activity, such as CD55 and CD59 shouldpreferably be removed from the composition. These surface glycoproteinsplay a role in inhibiting complement. CD55 destabilizes both C3convertases (C4b,2b and C3b,Bb) and both C5 convertases (C4b,2b,3b andC3b,Bb,C3b). CD59 and other homologous restriction factors arecell-surface expression proteins that inhibit the intercalation of C9into the plasma membrane, protecting blood cells, vascular endothelialcells, and other tissues from lytic damage by complement cascade.(Hoffman, 1999) CD55 and CD59 are membrane bound and preferably may beselectively removed by treatment with Phosphatidylinositol-specificPhospholipase C(PI-PLC). This will remove all of theglycosylphosphatidylinositol (GPI) linked proteins, including CD55 andCD59. Such selective enzyme cleavage is among known treatment methods.

Desialation of the inactivated composition is a beneficial step.Preferably, the sialic acid residue from HIV may be removed by treatmentwith neuramimidase enzyme. Neuramimidase cleaves sialic acid. (Hart,Melanie L., et al., AIDS Res Hum Retroviruses, Vol. 18(17), pp. 1311-7(2002)) (Meri, 1990) Many of the mannose sugar moieties are protectedfrom the immune system by a coating of sialic acid residue. Sialic acidis found on normal human cells and tissues, and is used by the immunesystem for “host recognition.” In doing so, the immunologic responseagainst “host” structures is limited. Sialic acid residues are alsofound on the heavily glycosylated envelope of HIV. (Hart, 2002) If thesialic acid residues are removed, then Factor H will not bind to HIV andthe vaccine would then be identified as “foreign” by the immune system,so that a robust immunologic response will occur. (Hart, 2002) Otherneutralization or desialation methods consistent with the presentinvention may also be used, such as treatment with trypsin.

In general, treatment of the structural features may be conducted at avariety of points in the preparation of the composition. Desialation ispreferable after exposure to psoralen and irradiation with ultravioletlight, in that desialation of active HIV can increase viral replicationand infectivity. (Hart, 2002)

Preparation for Administration

The present immunogenic composition may be mixed with appropriate immunestimulant or adjuvant, including those described as alternativeembodiments below. Such compositions may be used as are appropriate forthe application. Customary stimulants or adjuvant known in the artinclude incomplete Freund's adjuvant, liposomes, etc. A preferredembodiment includes one or more stimulant taken from customary adjuvantsand/or those compositions described further herein.

The immunogenic composition may also be mixed with pharmaceutical orlaboratory compositions appropriate for the anticipated method of use oradministration, such as carriers or excipients, such as mineral oil.

HIV Strains and Subtyping

As noted above, HIV has a diverse genotype. The high level of genomicdiversity of HIV complicates diagnosis and treatment of strains that canvary in infectivity, transmissibility, and immunogenicity. Theinvestigation of multivalent immunogenic compositions will necessarilyincorporate a plurality of strains. Phylogenetic analysis may beundertaken using resources from HIV sequence databases known to those inthe art, and as described herein.

The acquisition, isolation, and identification of HIV subtypes andsub-subtypes are well described in the field. (Robinson, D. L., et al.,HIV-1 Nomenclature Proposal: A Reference Guide to HIV-1 Classification(September 1999)) For the present invention, subtyping HIV may beperformed using any medically appropriate means known to those skilledin the art. Genotyping is a direct means for identifying HIV-1 subtypes.Serotyping or analysis of subtypes by antibody response of an infectedperson is an alternate, indirect means. DNA and RNA sequencing orgenotyping kits are preferable when they include phylogenetic analysisto detail the subtypes of concern. Subtype probes and assays of env andgag (heteroduplex mobility assays or HMAs) are alternative, but possiblylimited techniques. (Salminen, Mika, Nat'l Pub. Health Inst.(1994))(Buonaguro, Luigi, J. of Virology, Vol. 69, No. 12 pp. 7971-7981(1995)) In general, higher volume subtyping may be limited todiscriminating fewer subtypes.

The individual HIV-1 groups M (major), 0 (outlier) and N(non-major/outlier) may have arisen from separate transmission events;HIV-1 is somewhat similar to chimpanzee SIV. Phylogeneticdiversification within each of these groups into subtypes or cladesresulted from genetic mutations. Group M strains are primarilyresponsible for most HIV infections, while groups O and N strains areisolated largely from individuals from Cameroon.

The HIV-1 group M subtypes and sub-subtypes appear to have geneticallydiverged in humans from a common source. The members of group M arebroken out into nine equidistant phylogenetic subtypes. These arelabeled A1, A2, B, C, D, F1, F2, G, H, J and K. The sequences within anyone-subtype or sub-subtype share more genetically than they do withsequences from other subtypes throughout their genomes. Subtypesconstitute different lines of HIV, and may be statistically associatedwith social or geographic factors. Thus, the subtyping system identifiesgenetic clusters; it can be used for locating subtype specificbiological differences or similarities in HIV components, such asproteins. As an additional complication, viruses within a subtype mayevolve at different rates, while subtypes may also vary in rates ofevolution.

Retroviruses have a tendency to recombine with related retroviruses.This recombination is another contribution to genetic diversity. Theprocess of recombination creates variants, drug resistance, and modifiedexpression of antigenic characteristics. The circulating recombinantforms (CRF) of HIV are recombinant HIV-1 genomes that have made anepidemiologically relevant effect in the HIV-1 epidemic. CRFs of HIV arecharacterized by identical mosaic structures. Recombination can occurduring reverse transcription, when there is alternate switching oftemplates between both genomic RNA strands co-packaged in each of thevirions. If the two strains belong to different subtypes of the HIV-1 Mgroup, then the result is a mosaic genome composed of regions from eachof the two subtypes; that is, the viral reverse transcriptase enzymeswitches templates from one of packaged RNA genome to the other duringreverse transcription after the co-packaged genomes enter a new cell.

These inter subtype mosaics are commonly found in multiply-infectedindividual patients. If there is transmission of an inter-subtyperecombinant virus, then it can create a circulating strain within theHIV epidemic. CRFs are labeled with numbers in which they were firstdescribed. As of 2002, there were nearly 3 million CRF infections; CRFinfections are becoming more likely, adding to genomic diversity.

Although genetically and antigenically related, HIV-2 is biologicallyand epidemically distinct from HIV-1. Different groups of HIV-1 havebeen located throughout the world, while HIV-2 has been locatedpredominately in Africa, Europe, and India. HIV-2 is related to SIVisolated from the Sooty-mangabey. The sequences from the HIV-2 A, B, C,F or G clades are distinct from those in the D and E clades, leading tothe conclusion that each group of HIV-2 represents a separatetransmission event. HIV-2 appears to have lower infectivity andpathogenicity.

Description of Additional Embodiments

In an alternative embodiment, the composition may be bound covalently orotherwise to polysaccharides composed of mannose or mannan. Binding orcoupling may be accomplished using methods known to those in the field.Mannose is a sugar found only on microorganisms and pathogens notordinarily found within the human body. Mannose binding protein (MBP) isa colectin, a C-type lectin that contains regions of collagenousstructure. It is present in normal human serum and consists of subunitseach composed of three polypeptide chains, forming a collagen-liketriple helix and three C-terminal globular carbohydrate recognitiondomains (CRDs). Six subunits together form an overall structureresembling the bouquet of tulip-like structure of C1q of the classicalcomplement pathway. Binding of MBP to carbohydrate initiates theclassical complement pathway to the activation of C1r₂ C1s₂. This mayresult in complement killing either directly through insertion of theterminal membrane attack complex or through opsonization by depositionof complement on the microbial surface. MBP may also activate C2 and C4via another newly described serine proteases called MASP (1 and 2)serine proteases. Thus, MBP also exhibits complement independentopsonizing activity, probably mediated by binding of the collagenousstalks to the colectin receptor of phagocytic cells. (Prodinger, W. M.,et al., Fundamental Immunology, Ch. 29, pp. 967-95 (4th ed.1999))(Speth, Cornelia, et al., The Middle Eu. J. of Medicine, Vol. 111(10) pp 378-391 (1999)) Any organism with mannose or mannan on itssurface will stimulate the lectin pathway of complement activation. Acomposition bound to such polysaccharides will bind with mannose bindinglectin in the serum, activating the lectin pathway of the complementsystem. Thus, this alternative embodiment would thereby enhance theoverall immunologic response to the vaccine.

In another alternate embodiment, the composition may be combined withsubstances that stimulate or activate the alternative complementpathway. For example, it is known that certain forms of teichoic acidare potent activators of the alternative complement pathway.(Winkelstein, J. A., et al., J. of Immunol., Vol. 120(1), pp. 174-8(January 1978)) In addition, zymosan, which may be derived from yeastcells, can induce cytokines and stimulate immune response in conjunctionwith the alternative pathway of the complement system. Zymosan isphagocytosed by macrophages with or without opsonization, and thereforehas a useful immunologic property of activating the alternative pathwayof complementation. The zymosan macrophage interaction is believed toenhance the Th-1 response. CD4 cells can be divided into Th-1 and Th-2cells. Th-1 cells activate cytotoxic T cells by producing IL-2; whereasTh-2 cells form the B-cell helper function by producing primarily IL4and IL-5. The level of Th-1 response produced by zymosan is regulated byC3 cleavage fragments, C3b and iC3b. The amplified C3b deposits on theacceptor surface of zymosan and assembles macrophages, dendritic cells,or other antigen-presenting cells. Macrophages, dendritic cells, orother antigen-presenting cells make an antigen presentation to Th-1cells after opsonizing zymosan, and after antigen-specific macrophageactivation occurs. (Ara, Yuki, et al., Immun. Vol. 103(1), pp. 98-105(May 2001)) Zymosan can therefore be used as an immune stimulant; itenhances both humoral and cell-mediated immune responses to HIV disease.Thus, the composition may be bound covalently or otherwise to substancesthat stimulate the alternative complement pathway, such as teichoic acidor zymosan.

Therefore, to enhance immunogenicity, mannose, teichoic acid, zymosan,or some combination thereof may be bonded to protein components of thecomposition. Preferably, the polysaccharides may consist of sixteenseparate saccharide units. (Pangburn, M. K., 1989) The preferred sourcefor the carbohydrate/stimulant component of the composition would be thecapsular polysaccharide of the yeast cell, Cryptococcus neoformansserotype C. (Sahu, Arvind, et al., Biochem. J. Vol. 302, Part 2, pp.429-36 (Sep. 1, 1994)) This yeast cell exhibits four branching xylosesugars from each trimannose repeat unit. FIG. 2 demonstrates the loss ofactivity of Factors H and Factor 1 on C3b with increasing polysaccharidelength to a maximum of 16. If such yeast is likely to introduce DNArepair enzymes, then the enzymes are preferably to be inactivated usingthe methods described above.

Additionally glucose molecules and polysaccharides may be removed fromthe composition. Glucose inhibits both the rate and the extent of C3bdeposition. (Sahu, 1994) Glucose may be removed by adding insulin tocell cultures.

In an alternate embodiment, the effect of heparin may be inhibited.Heparin is a cofactor necessary for effective Factor H function.(Linhardt, Robert J., et al., Biology Chemistry, Vol. 263(26), pp.13090-6 (Sep. 18, 1988))(Maillet, Francoise, et al., Mol. Immun., Vol.25(9), pp. 917-23 (1998)) (Blackmore, 1996)(Blackmore, 1998) (Giannakis,Eleni, Immunopharmacology, Vol. 1(3), pp. 433-43 (October 2001)) FactorH is a major limiting protein in the alternative complement pathway. Thealternative complement pathway is the first arm of the immune system torespond to microorganisms or vaccines. Protamine binds heparin and isused to reduce the effective heparin in patients undergoinganticoagulation. (Maillet, Francoise, et al., Molecular Immun., Vol.20(12), pp. 1401-4 (December 1983)) (Weiler, John, et al., J. Exp. Med.,Vol. 147(2), pp. 409-21 (February 1978)) Recently, a less toxic heparinantagonist, low molecular weight protamine (LMWP) has become available.Protamine, or preferably LMWP for this embodiment, may be included as acomponent in order to impair the activity of Factor H in limiting thealternative complement pathway. (Liang, J. F., et al., Biochemistry,Vol. 68(1), pp. 116-20 (2003)) Alternatively, Heparinase is known todegrade Heparin enzymatically.

In another embodiment sulfated polyanions may be included in thevaccine. Sulfated polyanions absorb Factor H, effectively removing itfrom circulation. Other polyanions such as dextran sulfate and DNA areactivators of Factor H. These polyanions should be removed from thevaccine. Branched partially hydrolyzed polysaccharides of glucose knownas dextrans have been used for effective plasma expanders. (Hoffman,1999) Dextran sulfate is a sodium salt of sulfuric acid esters of thepolysaccharide dextran.

Soluble dextran sulfate with a molecular weight greater than5.times.10.sup.3 is an inducer of the alternative pathway of complement.The number of sulfate groups per hundred glucose residues in the dextrandetermined the activation potency of the dextran in the alternativepathway. The optimal degree of sulphation was 50-60 SO₄/100 glucosemolecules. (Burger, R., et al., Immunology, Vol. 29(3), pp. 549-54(1975))

Sulphated sephadex (SS) is a cross-linked insoluble form of dextran.Like soluble dextran sulphate SS activate the alternative pathway ofcomplement and the classical pathway as well. Three variables controlthe activity of SS with both pathways of complement activity. (1) Amountof sulphation; the higher the sulphated content up to 15.6% by weightresulted in a higher complement activation. No complement activation wasnoted with a sulphate content less than 2.43%. (2) Concentration of SS;higher concentrations result in complement activation with a maximum C3turnover at 40-50 μg/ml. (3) Temperature; maximum C3 turnover was notedat 37 degree C. with a total loss of activity at 4° C. (Burger, R., etal., Immunology, Vol. 33(6), pp. 827-37 (December 1977))

Both soluble and insoluble forms of dextran (>5000 molecular weight)activate the alternative pathway of complement. This is accomplished byblocking the effect of factor H. (Bitter-Suermann, D, et al., EuropeanJ. of Immun., Vol. 11(4), pp. 291-5 (April 1981))

Low molecular weight dextran sulfate (<5000) enhances factor H bindingtherefore it limits the activity of the alternative pathway ofcomplement. (Meri, 1990) DNA like heparin also increase factor Hbinding. (Gardner, William D., Biochemical and Biophysical ResearchCommunications, Vol. 94, pp 61-67 (1980))

Therefore to enhance immunogenicity dextran sulfate with a molecularweight>5000 with 50-60 SO₄/100 glucose molecules could be included inthe compound. Likewise SS with 15.6% SO₄ by weight at a concentration of40-50 μg/ml at a temperature of 37.degree. would enhance theimmunogenicity of the compound. Low molecular weight dextran would notbe included in the formulation since it would increase factor H bindingand decrease complement activation. Finally, DNA enhances complementactivity and therefore this immunogen could be used concurrently with aDNA vaccine. (The DPT vaccine is composed of three separate vaccineparticles. The pertussis component acts as an adjuvant for the othertwo. (Parham, Peter, The Immune System, Ch. 12 (2nd ed. 2004)) Ananalogous situation exists here, where a DNA vaccine for HIV diseasewould act as a adjuvant for the psoralen vaccine.)

In a further alternate embodiment, substances that stabilize C3convertase may be used with the present invention. All three complementpathways lead to the production of C3b, which bonds covalently to thesurface of microorganisms or components of the microorganisms presentedin such an immunogenic composition. C3b is produced by enzymes known asC3 convertase. Cobra venom factor (CVF), derived from the snake Najakaouthia, stabilizes this enzyme. (Alper, C. A. and D. Balavitch,Science, Vol. 191(4233), pp. 1275-6 (March 1976)) The half life of CVFC3b,Bb C3/C5 convertase is seven hours, in contrast to that ofendogenously produced alternative complement pathway C3 convertase(C3b,Bb), which is 1.5 minutes. C3b,Bb is disassembled by Factor H andC3b is inactivated by the combined action of Factor H and Factor I. Incontrast Factor CVF,C3,Bb is resistant to all regulatory complementproteins. (Kock, Michael A., et al., J. of Biol. Chemistry, Vol.279(29), pp. 30836-43 (July 2004)) C3b,Bb requires additional C3b to acton C5 whereas CVF,Bb can cleave C5 directly. Therefore, the CVF,Bbenzyme continuously activates C3 and C5. (Kock, 2004)

The biological function of CVF in cobra venom is believed to facilitatethe entry of the toxic venom components into the bloodstream. This isachieved by complement activation causing release of the anaphylatoxinsC3a, C5a and Bb, which increase the vascular permeability. (Vogel, Carl,Immunoconjugates, Ch. 9 (1987)) CVF, despite its derivation from cobravenom, is a non-toxic protein; CVF can be isolated from the otherenzymes, polypeptides, etc., from cobra venom, which includes toxins.

Thus, administration of CVF results in an explosive production of C3b.(Vogel, 1987) (Kock, 2004) FIG. 3 illustrates the structural homologybetween C3 and CVF. C3b on the surface of microorganisms is recognizedby follicular dendritic cells within the lymph nodes as well as T cellsand B cells in the peripheral circulation and within the germinalcenters of the lymph nodes. C3b is a powerful opsonin. Opsonins triggerseveral arms of the immune system simultaneously. (Hoffman, 1999) Thus,in an alternatively embodiment, CVF may be used as a component of thecomposition.

The preferred form of CVF is dCVF (De-α-galactosylated CVF). (Gowda, D.C., et al., J. of Immunology, Vol. 152(6), pp. 2977-86 (March 1994))Naturally occurring CVF is characterized by an unusual polysaccharidewhich is a fucosylated biantennary complex-type N-linked chaincontaining an α-galactosylated Lex antigenic epitope, Galα1-3Galβ1-4(Fucα1-3) GlcNAcβ1. Removal of this polysaccharide can be accomplishedby incubating CVF with peptide-N-glycosidase F (N-glycanase) at 37° C.for 18 to 23 hours at a ph of 8.0. Removal of this novel polysaccharidefrom CVF is necessary since 1% of human IgG reacts with the terminalGalα1-3Galβ1 sequence of CVF. However removal of this polysaccharidedoes not interfere with the complement fixation character of themolecule nor does it result in a shorter half life of the molecule. dCVFwill be covalently bound to the polysaccharide unit(s) comprising theimmunogenic composition.

In another embodiment, nickel compounds may be added to the composition.It has been shown that nickel is effective in enhancing the C3convertase activity of both the lectin and the alternative complementpathways. (Fishelson, Z., et al., J. of Immun., Vol. 129(6), pp. 2603-7(December 1982)) Natural nickel intake for average adults is estimatedto be 60 to 260 micrograms per day, with an environmental healthreference dose of 0.02 milligram per kilogram body weight per day(mg/kg/d). (U.S. EPA, 2003) It is contemplated that the presentinvention would include Nickel, preferably nickel chloride, on the orderof average daily intake well below the reference dose. Therefore, thepresent invention may be produced using nickel to enhance immuneresponse.

SUMMARY

In conclusion, the present invention is an immunogenic composition and awhole particle vaccine based on psoralen inactivation of predeterminedstrains of HIV, wherein certain features that interfere with immuneresponse have been treated. Individual or component vaccines may becreated for HIV subtypes or circulating recombinant forms of concern, aslisted above or as may be identified in the future.

The process for determining what strain of HIV should be included in theimmunogenic composition depends on the contemplated application. For atherapeutic example, a PBMC specimen may be drawn from an HIV positivepatient; and from this sample appropriate regions (e.g., env gp41, gagp24) of the HIV genome may be isolated, amplified and sequenced todetermine the HIV subtype. Phylogenetic analysis may be undertaken usingresources from HIV sequence databases. At the same time, viral loadingmay be assessed.

A methodology of the present invention comprises for preparation of animmunogenic composition includes:

1. Determining and isolating the strains of HIV that are of concern

2. Culturing the strains, optionally in a DNA repair enzyme deficientculture

3. Separating the virus from the culture media

4. Optionally removing the cellular outer plasma membrane

5. Adding psoralen and optionally a DNA-repair enzyme blocking agent

6. Irradiating with ultraviolet light

7. Removing or neutralizing CD55 and CD59

8. Desialation of the inactivated virus

9. Optionally adding appropriate immune stimulants or adjuvants

To prepare the composition that constitutes the composite vaccine agentfor the invention, it is possible to use known methods of purification,synthesis, or genetic engineering for each of the components.Practitioners skilled in the art may isolate and inactivate viralstrains in the preparation of the vaccines. These may be incorporatedinto pharmaceutical compositions appropriate for the anticipated methodof administration, such as carriers or excipients. A patient requiringtreatment may be administered the present invention in amountssufficient to ameliorate immune response; that is, a therapeuticallyeffective dose would be that amount necessary to reverse specific immunesuppression in an HIV positive patient to the extent desired, anddetermined using standard means, such as Chromium Release Assay,Intracellular Cytokine Assay, Lympho-proliferative Assay (LPA),Interfero'n Gamma (IFN-gamma) ELISpot Assays, and preferably MHCTetramer Binding Assays. These same laboratory tests would be applied tomeasure the immune response of an HIV negative patient. Thetherapeutically efficacious or effective dosing and dosing schedulewould depend on the patient's age, gender, and co-morbid diseases.Furthermore, the potential for pregnancy is a factor in treatment ofwomen of child bearing potential.

The analysis and development of the immunogenic composition shouldincorporate a wide range of doses of inactivated particulate forevaluation. Animal trials should consider differences in size, species,and immunological characteristics; it is anticipated that immunologicaldifferences between humans and animals may relegate animal trials totoxicity analysis. Clinical trials will involve at least the standardthree phase model, ranging from safety and dosage in a small population,safety and immunogenicity in a second phase of several hundredvolunteers, to a large scale effectiveness phase. A starting dose fortrials may be 10 micrograms/strain for juveniles and 20micrograms/strain for adults. Testing should contemplate particulateconcentration in the wide range of 10-10 ²⁰. The clinical trials shouldinclude appropriate exclusionary criteria as is customary, such asexclusion for other immune suppression conditions, pregnancy, activedrug use, etc.

Administration may be made in a variety of routes, for example orally,transbucally, transmucosally, sublingually, nasally, rectally,vaginally, intraocularly, intramuscularly, intralymphatically,intravenously, subcutaneously, transdermally, intradermally, intratumor, topically, transpulmonarily, by inhalation, by injection, or byimplantation. etc. Various forms of the composition may include, withoutlimitation, capsule, gelcap, tablet, enteric capsule, encapsulatedparticle, powder, suppository, injection, ointment, cream, implant,patch, liquid, inhalant, or spray, systemic, topical, or other oralmedia, solutions, suspensions, infusion, etc. In addition, the presentinvention may be combined with other therapeutic agents, such ascytokines, including natural, recombinant and mutated forms, fragments,fusion proteins, and other analogues and derivatives of the cytokines,mixtures, other biologically active agents and formulation additives,etc. Those skilled in the art will recognize that for injection,formulation in aqueous solutions, such as Ringer's solution or a salinebuffer may be appropriate. Liposomes, emulsions, and solvents are otherexamples of delivery vehicles. Oral administration would requirecarriers suitable for capsules, tablets, liquids, pills, etc, such assucrose, cellulose, etc. Because some of the first targets for infectionwith HIV are epithelial cells and Langerhans cells in the skin andrectal and vaginal mucosa, then a preferable embodiment of delivery isdermal combined with rectal and/or vaginal suppository. HIV iscontracted predominantly by rectal and vaginal intercourse. Thereforerectal and/or vaginal suppository administration of the vaccine would bethe preferred administration methodology. The present invention may alsobe administered in a prime-boost protocol.

While the description above refers to particular embodiments of thepresent invention, it will be understood that many modifications may bemade without departing from the spirit thereof. The accompanying claimsare intended to cover such modifications as would fall within the truescope and spirit of the present invention.

1. (canceled)
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 3. (canceled)
 4. (canceled)
 5. (canceled) 6:A method of developing an immune response, comprising administering acomposition comprising an inactivated HIV virus and a pharmaceuticallyacceptable carrier, wherein the virus has been inactivated by exposureto ultraviolet radiation and psoralen, lacks CD55 and CD59 in the viralmembrane and has been subjected to desialation. 7: The method accordingto claim 6, wherein CD55 and CD59 are removed by treatment withphosphatidylinositol-specific phospholipase. 8: The method according toclaim 6, wherein sialic acid is removed by treatment with neuraminidase,trypsin, or other appropriate desialation enzymes. 9: The methodaccording to claim 6, wherein CD55 and CD59 are removed by treatmentwith phosphatidylinositol-specific phospholipase and wherein sialic acidis removed by treatment with neuraminidase. 10: The method according toclaim 6, comprising the additional step of removing from the compositionpolyanions capable of potentiating Factor H. 11: The method according toclaim 6, comprising the additional step of adding to the compositionsulfated polyanions capable of absorbing Factor H. 12: The methodaccording to claim 6, comprising the additional step of adding an immunestimulant to the composition. 13: The method according to claim 12,wherein the immune stimulant comprises polysaccharides composed of atleast one mannose in a form capable of binding to the composition. 14:The method according to claim 12, wherein the immune stimulant comprisesteichoic acid in a form capable of binding to the composition. 15: Themethod according to claim 12, wherein the immune stimulant compriseszymosan in a form capable of binding to the composition. 16: The methodaccording to claim 12, wherein the immune stimulant comprisescryptococcus neoformans serotype C having a polysaccharide capsulecapable of binding to the composition. 17: The method according to claim12, wherein the immune stimulant comprises protamine in a form capableof binding to heparin. 18: The method according to claim 12, wherein theimmune stimulant comprises a heparinase. 19: The method according toclaim 12, wherein the immune stimulant comprises cobra venom factor in aform adapted to enhance production of C3b. 20: The method according toclaim 19, wherein the cobra venom factor is dCVF. 21: The methodaccording to claim 12, wherein the immune stimulant comprises Nickel ina form adapted to enhance C3 convertase activity. 22: The methodaccording to claim 6, wherein administration of the composition is bycapsule, gelcap, tablet, enteric capsule, encapsulated particle, powder,suppository, injection, ointment, cream, implant, patch, liquid,inhalant, or spray.
 23. The method according to claim 6, whereinadministration of the composition is orally, transbucally,transmucosally, sublingually, nasally, rectally, vaginally,intraocularly, intramuscularly, intralymphatically, intravenously,subcutaneously, transdermally, intradermally, intra tumor, topically,transpulmonarily, by inhalation, by injection, or by implantation.